vanA Gene Cluster Research Articles

SHIN-2 exerts potent activity against VanA-type vancomycin-resistant Enterococcus faecium in vitro by stabilizing the active site loop of serine hydroxymethyltransferase

Novel classes of antibiotics are needed to improve the resilience of the healthcare system to antimicrobial resistance (AMR), including vancomycin resistance. vanA gene cluster is a cause of vancomycin resistance. This gene cluster is transferred and spreads vancomycin resistance from Enterococcus spp. to Staphylococcus aureus. Therefore, novel antibacterial agents are required to combat AMR, including vanA-type vancomycin resistance. Serine hydroxymethyltransferase (SHMT) is a key target of antibacterial agents. However, the specific binding mechanisms of SHMT inhibitors remain unclear. Detailed structural information will contribute to understanding these mechanisms. In this study, we found that (+)–SHIN–2, the first in vivo active inhibitor of human SHMT, is strongly bound to the Enterococcus faecium SHMT (efmSHMT). Comparison of the crystal structures of apo- and (+)–SHIN–2-boud efmSHMT revealed that (+)–SHIN–2 stabilized the active site loop of efmSHMT via hydrogen bonds, which are critical for efmSHMT inhibition. Additionally, (+)–SHIN–2 formed hydrogen bonds with serine, forming the Schiff's base with pyridoxal 5′-phosphate, which is a co-factor of SHMT. Furthermore, (+)–SHIN–2 exerted biostatic effects on vancomycin-susceptible and vanA-type vancomycin-resistant E. faecium in vitro, indicating that SHMT inhibitors do not induce cross-resistance to vanA-type vancomycin. Overall, these findings can aid in the design of novel SHMT inhibitors to combat AMR, including vancomycin resistance.

Prevalence and Molecular Characterization of Vancomycin Variable Enterococcus faecium Isolated From Clinical Specimens.

Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole-genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control.

The population structure of vancomycin-resistant and -susceptible Enterococcus faecium in a low-prevalence antimicrobial resistance setting is highly influenced by circulating global hospital-associated clones.

Between 2010 and 2015 the incidence of vancomycin-resistant Enterococcus faecium (VREfm) in Norway increased dramatically. Hence, we selected (1) a random subset of vancomycin-resistant enterococci (VRE) from the Norwegian Surveillance System for Communicable Diseases (2010-15; n=239) and (2) Norwegian vancomycin-susceptible E. faecium (VSEfm) bacteraemia isolates from the national surveillance system for antimicrobial resistance in microbes (2008 and 2014; n=261) for further analysis. Whole-genome sequences were collected for population structure, van gene cluster, mobile genetic element and virulome analysis, as well as antimicrobial susceptibility testing. Comparative genomic and phylogeographical analyses were performed with complete genomes of global E. faecium strains from the National Center for Biotechnology Information (NCBI) (1946-2022; n=272). All Norwegian VREfm and most of the VSEfm clustered with global hospital-associated sequence types (STs) in the phylogenetic subclade A1. The vanB2 subtype carried by chromosomal Tn1549 integrative conjugative elements was the dominant van type. The major Norwegian VREfm cluster types (CTs) were in accordance with concurrent European CTs. The dominant vanB-type VREfm CTs, ST192-CT3/26 and ST117-CT24, were mostly linked to a single hospital in Norway where the clones spread after independent chromosomal acquisition of Tn1549. The less prevalent vanA VRE were associated with more diverse CTs and vanA carrying Inc18 or RepA_N plasmids with toxin-antitoxin systems. Only 5 % of the Norwegian VRE were Enterococcus faecalis, all of which contained vanB. The Norwegian VREfm and VSEfm isolates harboured CT-specific virulence factor (VF) profiles supporting biofilm formation and colonization. The dominant VREfm CTs in general hosted more virulence determinants than VSEfm. The phylogenetic clade B VSEfm isolates (n=21), recently classified as Enterococcus lactis, harboured fewer VFs than E. faecium in general, and particularly subclade A1 isolates. In conclusion, the population structure of Norwegian E. faecium isolates mirrors the globally prevalent clones and particularly concurrent European VREfm/VSEfm CTs. Novel chromosomal acquisition of vanB2 on Tn1549 from the gut microbiota, however, formed a single major hospital VREfm outbreak. Dominant VREfm CTs contained more VFs than VSEfm.

Isolation of a Multidrug-Resistant vanA-Positive Enterococcus faecium Strain from a Canine Clinical Sample in Greece

An Enterococcus faecium strain was obtained from a paraprostatic cyst of a 17-year-old dog in Greece. Antibiotic susceptibility testing (AST) was accomplished by disc diffusion and MIC methods, and the isolate demonstrated a multidrug-resistant (MDR) phenotype against a great variety of antibiotics, such as β-Lactams, Quinolones, Macrolides, Tetracyclines, Rifampin, Nitrofurantoin, and surprisingly, Glycopeptides, Fosfomycin and Gentamicin (high-level). Molecular screening for Vancomycin resistance genes was carried out, and a vanA gene cluster was identified. To our knowledge, this is the first report of a vanA-positive E. faecium strain isolated from a companion animal in Greece. Importantly, this strain was related with the presence of paraprostatic cysts, a pathological condition requiring treatment. The presence of a highly resistant isolate in a canine clinical sample and the consequent need for treatment constitutes a new challenge for veterinarians due to the lack of available treatment options. Our findings indicate the occurrence of respective bacteria in companion animals, which could act as a reservoir of epidemic MDR strains or relevant mobile genetic elements (MGE) in the community, constituting a threat for public health.

Genomic Analysis of Vancomycin-Resistant Staphylococcus aureus Isolates from the 3rd Case Identified in the United States Reveals Chromosomal Integration of the vanA Locus.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a human pathogen of significant public health concern. Although the genome sequences of individual VRSA isolates have been published over the years, very little is known about the genetic changes of VRSA within a patient over time. A total of 11 VRSA, 3 vancomycin-resistant enterococci (VRE), and 4 methicillin-resistant S. aureus (MRSA) isolates, collected over a period of 4.5 months in 2004 from a patient in a long-term-care facility in New York State, were sequenced. A combination of long- and short-read sequencing technologies was used to obtain closed assemblies for chromosomes and plasmids. Our results indicate that a VRSA isolate emerged as the result of the transfer of a multidrug resistance plasmid from a coinfecting VRE to an MRSA isolate. The plasmid then integrated into the chromosome via homologous recombination mediated between two regions derived from remnants of transposon Tn5405. Once integrated, the plasmid underwent further reorganization in one isolate, while two others lost the staphylococcal cassette chromosome mec element (SCCmec) determinant that confers methicillin-resistance. The results presented here explain how a few recombination events can lead to multiple pulsed-field gel electrophoresis (PFGE) patterns that could be mistaken for vastly different strains. A vanA gene cluster that is located on a multidrug resistance plasmid that is integrated into the chromosome could result in the continuous propagation of resistance, even in the absence of selective pressure from antibiotics. The genome comparison presented here sheds light on the emergence and evolution of VRSA within a single patient that will enhance our understanding VRSA genetics. IMPORTANCE High-level vancomycin-resistant Staphylococcus aureus (VRSA) began to emerge in the United States in 2002 and has since then been reported worldwide. Our study reports the closed genome sequences of multiple VRSA isolates obtained in 2004 from a single patient in New York State. Our results show that the vanA resistance locus is located on a mosaic plasmid that confers resistance to multiple antibiotics. In some isolates, this plasmid integrated into the chromosome via homologous recombination between two ant(6)-sat4-aph(3') antibiotic resistance loci. This is, to our knowledge, the first report of a chromosomal vanA locus in VRSA; the effect of this integration event on MIC values and plasmid stability in the absence of antibiotic selection remains poorly understood. These findings highlight the need for a better understanding of the genetics of the vanA locus and plasmid maintenance in S. aureus to address the increase of vancomycin resistance in the health care setting.

Acquisition of Daptomycin Resistance by Enterococcus faecium Confers Collateral Sensitivity to Glycopeptides.

Daptomycin is a last-line antibiotic used in the treatment of multidrug-resistant Enterococcus faecium infections. Alarmingly, daptomycin-resistant E. faecium isolates have emerged. In this study, we investigated the evolution and mechanisms of daptomycin resistance in clinical E. faecium isolates and the corresponding acquisition of collateral sensitivity (CS) as an evolutionary trade-off. We evolved daptomycin resistance in six daptomycin-susceptible E. faecium isolates to obtain daptomycin-resistant mutants. The six E. faecium strains successfully acquired high-level resistance to daptomycin in vitro, but this led to fitness costs in terms of growth, in vitro competition, and virulence. Mutations in liaFSR, yycFG, and cls; increased surface positive charge; thicker cell walls; and elevated expression of dltABCD and tagGH were observed in daptomycin-resistant mutants. Surprisingly, we observed the emergence of CS in SC1762 isolates after the induction of daptomycin resistance. Compared with parental strains, the SC1174-D strain (i.e., daptomycin-resistant mutant of SC1174; non-CS) showed significantly upregulated expression of the vanA gene cluster. However, in SC1762-D (i.e., daptomycin-resistant mutant of SC1762), all vanA cluster genes except the vanX gene were obviously downregulated. Further in silico analyses revealed that an IS1216E-based composite transposon was generated in SC1762-D, and it disrupted the vanH gene, likely affecting the structure and expression of the vanA gene cluster and resulting in resensitization to glycopeptides. Overall, this study reports a novel form of CS between daptomycin and glycopeptides in E. faecium. Further, it provides a valuable foundation for developing effective regimens and sequential combinations of daptomycin and glycopeptides against E. faecium.

Collateral sensitivity to pleuromutilins in vancomycin-resistant Enterococcus faecium

The acquisition of resistance to one antibiotic sometimes leads to collateral sensitivity to a second antibiotic. Here, we show that vancomycin resistance in Enterococcus faecium is associated with a remarkable increase in susceptibility to pleuromutilin antibiotics (such as lefamulin), which target the bacterial ribosome. The trade-off between vancomycin and pleuromutilins is mediated by epistasis between the van gene cluster and msrC, encoding an ABC-F protein that protects bacterial ribosomes from antibiotic targeting. In mouse models of vancomycin-resistant E. faecium colonization and septicemia, pleuromutilin treatment reduces colonization and improves survival more effectively than standard therapy (linezolid). Our findings suggest that pleuromutilins may be useful for the treatment of vancomycin-resistant E. faecium infections.

Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions.

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration.

Identification of Vancomycin Resistance Determinants in Twin Cities of Rawalpindi/Islamabad

Vancomycin resistant Enterococci have emerged rapidly in the recent years leading to treatment failure. The aim of this work was to identify vancomycin resistance determinants; vanA and vanB genes in Enterococci. The blood, urine and throat samples were collected from 150 patients from local hospitals of twin cities of Rawalpindi/Islamabad. Forty-nine phenotypically confirmed isolates were further confirmed by PCR amplification for vancomycin determinants (genes for vanA and vanB) for vancomycin resistant Enterococci. Dependence and frequency distribution of VRE and VSE bacteraemia with respect to age, gender and source was also studied. Phenotypically resistant strains were positive for vanA while negative for vanB. vancomycin susceptible enterococci (VSE) could be isolated more from urine samples as compared to blood whereas vancomycin resistant enterococci (VRE) was found more prevalent in the blood samples (p value= 0.000). VRE was more frequently isolated from patients aged 50 or above whereas VSE prevalence was same in both age groups (p value=0.002). Gender was not found to have any significant impact on VRE or VSE bacteraemia. This study reports vanA gene cluster responsible for resistance in Pakistani population and frequently isolation of VRE from blood samples. Keywords: Enterococcus, Vancomycin resistant Enterococci, Pakistan

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

Spread of vancomycin-resistant Enterococcus faecium ST133 in the aquatic environment in Switzerland

The global dissemination of vancomycin-resistant enterococci (VRE) has become a serious public-health concern. Although outbreaks are typically caused by nosocomial transmission, contaminated food and water may contribute to the spread of VRE. The aim of this study was to assess the presence of VRE in flowing surface water bodies in Switzerland and to characterise the isolates. Surface water was sampled from rivers, streams and canals throughout Switzerland and was screened for the presence of VRE. Whole-genome sequencing was used to identify antimicrobial resistance genes and the phylogenetic similarity of the obtained isolates. VRE were detected in 6 (3.1%) of 191 water samples. The six VRE-containing samples were all collected near treated wastewater discharge sites. The six isolates were identified as Enterococcus faecium sequence type 133 (ST133) and harboured the vancomycin resistance-conferring vanA gene cluster on transposon Tn1546. They showed a close phylogenetic relationship to ST133 swine faecal isolates obtained during a previously reported screening in Switzerland. Our results suggest that surface water contributes to the environmental dissemination of VRE. Repeated identification of ST133 clones in geographically distinct water sampling sites and swine faecal samples collected in slaughterhouses may indicate a local dominance of this VRE lineage in Switzerland.

Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

BackgroundEnterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations.MethodsWe performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination.ResultsOn average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination.ConclusionsIn related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture.

To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

First Report of the Local Spread of Vancomycin-Resistant Enterococci Ascribed to the Interspecies Transmission of a vanA Gene Cluster-Carrying Linear Plasmid.

Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid.IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.

Expression of VanA-type vancomycin resistance in a clinical isolate of Enterococcus faecium showing insertion of IS19 in the vanS gene

The characteristics of an unusual clinical isolate of Enterococcus faecium (CL-6729) showing insertion of IS19 (also known as ISEfm1) in the vanS gene while maintaining a constitutive VanA phenotype are described. This isolate was obtained from a hospital-acquired urinary tract infection, showed multidrug resistance by antimicrobial susceptibility testing, and belongs to ST78 based on multilocus sequence typing (MLST). Except for the vanS gene, all the other genes of the vanA gene cluster were intact according to conventional PCR, overlapping PCR and genome sequencing. By quantitative reverse transcription PCR (RT-qPCR), the isolate showed similar expression of the vanA, vanR and vanS genes in the presence and absence of vancomycin. The results suggest that insertion of IS19 in the vanS gene may be associated with constitutive expression of resistance to vancomycin in clinical isolate CL-6729, either by not impairing VanS activity or by inducing the emergence of another pathway that acts on vanA expression, which still needs to be fully investigated.

Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate.

Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5′ end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.

622. The Accessory Genome in Enterococcal Bacteremia: Results from the Vancomycin-Resistant Enterococcal Bacteremia Outcomes Study (VENOUS)

BackgroundVancomycin-resistant enterococci (VRE) are a major cause of nosocomial bloodstream infections. Enterococci exhibit remarkable genomic plasticity and can recombine through the acquisition of genetic material via mobile genetic elements (MGEs), including resistance genes. The accessory genome plays a major role in the evolution of enterococci within the human host. Thus, dissecting the entire genome (pan-genome) is of paramount importance to characterize the population structure of enterococci causing disease.MethodsVENOUS is an ongoing prospective, observational study of adults with enterococcal bacteremia. From September 2016 to March 2018, E. faecalis (Efs) and E. faecium (Efm) were collected in 14 hospitals of a single hospital system and a major cancer center in Houston, TX, and a general hospital in Detroit, MI. Short- and long-read genomic sequencing were performed with Illumina MiSeq and Oxford Nanopore Technologies GridION X5, respectively. A proprietary bioinformatics pipeline was utilized for genome assembly and further analyses.Results156 Efs and 98 Efm isolates from single patients were analyzed. The average proportion of core genes in each genome was 64.6% (53.0–74.1) and 49.1% (45.2–51.0) for Efs and Efm, respectively. The vanA gene cluster was identified in 5.1% (8/157) of Efs and 57.1% (56/98) of Efm. The plasmid-encoded aac(6′)-Ie-aph(2″)-Ia gene conferring high-level resistance to aminoglycosides was found in 37.6% (59/157) Efs, seven of which also possessed vanA. Long-read sequencing of vanA-harboring plasmids from a subset of VRE revealed that the vanA cluster was carried in plasmids ranging from 31.7 to 132.3 kb. Although the vanA operon was fairly conserved, insertions of MGE were identified in the intergenic regions of vanS/vanH and vanX/vanY. Furthermore, a variety of MGE insertions mediated integration of the vanA operon, including IS1216 and IS256 (figure).ConclusionAccessory genes, including AMR genes, comprise a significant proportion of the enterococcal pan-genome, indicating major genetic plasticity within these organisms. Acquired resistance genes seem to have a high degree of recombination and play a substantial role in the expansion of the genomic repertoire in clinical isolates. DisclosuresSamuel L. Aitken, PharmD, Melinta Therapeutics: Grant/Research Support, Research Grant; Merck, Sharpe, and Dohme: Advisory Board; Shionogi: Advisory Board.

Whole-genome sequencing revealed independent emergence of vancomycin-resistant Enterococcus faecium causing sequential outbreaks over 3years in a tertiary care hospital.

Vancomycin-resistant Enterococcus faecium (VREfm) emerged as an important cause of nosocomial infections worldwide. Previous studies based on molecular typing revealed that VREfm outbreaks are mainly associated with a particular genetic lineage, namely clonal complex 17 (CC17), which harbours either vanA or vanB gene cluster. The University Hospital of Lausanne faced several VREfm episodes of transmissions between 2014 and 2017. In this study, we used whole-genome sequencing (WGS) to investigate the relatedness of 183 VREfm isolates collected from 156 patients. Sequence types (ST) 17, ST80 and ST117 were the most predominant clones. Based on epidemiological data, 10 outbreaks were identified, which were caused by at least 13 distinct genotypes. The majority of isolates involved in outbreaks (91%) differed by only 0 to 3 SNPs. Four outbreaks involved more than one genotype and half of the cases considered as sporadic were possibly linked to an outbreak. By sequencing all isolates, we were able to better understand our local epidemiology of VREfm. The polyclonal structure observed between the different outbreaks strains, the high level of recombination detected in isolates, the time elapsed between admission and the first VREfm detection and the negative screening at admission support the hypothesis of the emergence of new VREfm clones within the hospitalised population.

Enterococcal Infections and Antimicrobial Resistance in a Tertiary Care Hospital, Eastern India

Background and study aim: During last two decades, there has been a world-wide trend in increasing occurrence of entero-coccal infections in the hospitals. The aim of present study was to determine the spectrum of enterococcal infections, speciesprevalence, antimicrobial resistance and characteristics of vancomycin resistant enterococci (VRE) in a tertiary care hospital, Eastern India. Patients and Methods: Between January 2013 and July 2014, 152 Enterococcus species were obtained from clinical samples. Enterococci were identified using standard biochemical tests. Antimicrobial susceptibility was tested by Kirby-Bauer disk diffusion according to Clinical & Laboratory Standards Institute (CLSI) guidelines.VRE agar base was used to screen VRE isolates. Minimum inhibitory concentration (MIC) values of VRE isolates were determined using Epsilometer-test. VRE isolates were also examined by PCR to detect vanA gene. Results: From 1602 clinical samples, 961 (60%) were culture positive and 152(15.8%) enterococcal isolates were obtained. Most common species isolated was E. faecalis (63.8%) followed by E. faecium (35.5%). Majority of enterococcal infections were detected from ICUs and surgical wards and clinically presented as UTIs. Disk diffusion method showed 67.1% were resistant to penicillin, 61.2% ampicillin, 58.5% ciprofloxacin, 46.7% high-level gentamicin, 42. 8% high-level streptomycin, 7.9% teicoplanin and none to linezolid. Twenty (13.2%) enterococcal isolates were vancomycin resistant in VRE screen and disk diffusion method. Epsilometer-test of VRE isolates showed 8 (40%) isolates were resistant and 9 (45%) were intermediately resistant. From 20 VRE isolates, six showed VanA and two VanB phenotypes and all six VanA phenotypes had vanA gene cluster. Conclusion: More accurate and reliable MIC determination tests should be performedin all suspected VRE isolates. Confirmatory PCR is required for identifying resistant gene cluster.